1. Field of the Invention
The present invention relates to a method for detecting microorganisms which may be present in or on any source desired to be tested for the presence of microorganisms. More particularly, the present invention relates to a method of detecting the presence of microorganisms by depositing a source of microorganisms in a cell containing two electrodes, one of which is protected from exposure to the microorganisms and a nutrient medium and detecting the change in potential between the measuring electrode exposed to the growing microorganism and the reference electrode.
2. Description of the Prior Art
Presently, several methods are known for the detection of microorganisms which may be present in various sources which include aqueous media such as blood, plasma, fermentation media and the like. The methods are generally divided into two classes of detection in which the first is a screening test to determine whether or not large numbers of microorganisms are present in a sample. If a positive test is obtained by the first step, a second level of testing is employed to determine the type and amount of organism present. Most commonly, methods which establish both the identity and amount of microorganism are based upon a sequence of steps of culturing, growth and observation of the microorganism. Growth rates are observed in the culture which are derived from multiple dilutions of the same sample. By observing the time at which the diluted samples reach observable populations, the concentration of microorganisms in the original samples can be estimated.
The estimation of microorganism populations is most generally accomplished by one of three techniques. The first technique is a nutrient agar plating technique in which a microorganism is allowed to grow on an agar nutrient substrate and the growth of the microorganism is initially observed visually and thereafter by microscopic observation. This is the most common method in use clinically.
The second type of technique includes several methods which can be classified as chemical methods. One method of analysis involves supplying a microorganism in a growth medium with carbon-14 labeled glucose. The microorganism metabolizes the radioactive glucose and evolves C.sup.14 O.sub.2, which is sampled and counted. While positive results can be obtained by this method in a relatively short period of time, the method is encumbered by various operational complexities, is expensive and is hazardous from the standpoint of the necessity of handling radioactive samples. Another analytical method of determing the type and amount of bacteria present in a sample is based upon the chemiluminescent reaction between luciferase and luciferin in the presence of ATP. Bacteria are grown on a culture and then the cells are lyzed to free the ATP present therein. The liberated ATP reacts with the luciferase-luciferin combination whereby chemiluminescent light is emitted which is detected by a photomultiplier and used to determine the amount of the organism present. The principal disadvantage of this technique is the expense of the materials involved in the reaction. Still another chemical method involves a measurement based upon the metabolic conversion of nitrite ion to nitrate ion. This method, however, is only applicable to some bacteria and yeasts.
The third type of technique used for the detection of microorganisms involve non-chemical methods. One method involves the evaluation of relatively clear microorganism suspensions by modified particle counters. However, the method is non-specific and does not provide a distinction between viable microorganisms and dead ones, or even non-biologic particulate matter. A second method involves the detection of microorganisms by impressing an alternating current across a pair of electrodes which have been placed in a microorganism containing medium and observing the change in impedance of the current as a function of the growth of the microorganism. This technique however, is apparently not as amenable to automation as has been expected for the device and method.
A bio-chemical sensor is known as disclosed by Rohrback et al U.S. Pat. No. 3,403,081, which is used to detect trace elements and poisons in liquid and gaseous media. The sensor is constructed by placing a measuring electrode such as an inert wire gauge cylinder of nickel, platinum, stainless steel or the like upon which is impregnated a colony of a microorganism or an enzyme and a reference electrode such as the standard Calomel electrode in an electrolyte. The leads from each electrode are attached to a voltmeter. The organism or enzyme impregnated or in close proximity to the measuring electrode generates a current within the cell by causing chemical reactions at the surface of the electrode or by promoting chemical reactions to produce materials which in turn provide depolarization reactions at the electrode. The device functions by admitting a trace element or a poison into the device which deactivates the enzyme or kills or deactivates the microorganism thus causing a change in the potential difference between the electrodes which is detected by a change in the voltmeter readings. Considering the fact that the enzyme or microorganism promotes or causes a considerable chemical reaction at the measuring electrode which is detected by a standard voltmeter in the circuitry of the cell, the current generated within the cell must be substantial. The electroanalytical device of the present invention which is used to determine the type and amount of a microorganism in a solution, on the other hand, does not require the impregnation of a sizable colony of a microorganism on the measuring electrode but rather operates by detecting a microorganism in solution which gradually concentrates about the measuring electrode. A further critical distinction between the method and apparatus of the present invention and the method of the reference is that the circuitry of the present system must contain a high impedance potentiometer and not the conventional voltmeter used in the reference's process, because the present system is dependent upon the measurement of an electrostatic-like potential difference between the measuring electrode and the microorganism in solution. If a standard voltmeter were used in the circuitry of the present invention, far too much current would be drawn by the voltmeter which would destroy the relatively delicate electrostatic-like potential difference between the measuring electrode and the microorganism concentrated about the electrode's surface.
Consequently, a need continues to exist for a method of rapidly, automatically and economically determining types and amounts of various microorganism by a conceptually simple and economic technique.